PROJECT ABSTRACT The nuclear lamina is physically connected through nuclear envelope proteins to the cytoskeleton by the LINC complex (linker of nucleoskeleton to cytoskeleton), which spans the nuclear envelope and allows the transmission of mechanical forces to the nucleus. LINC complex proteins are frequently mutated or dysregulated in cancer, and some of these mutations have been proposed to be cancer drivers. Yet, how alterations to the LINC complex might promote cancer development is not known. This application's overarching hypothesis is that cytoskeletal force transmission to the nucleus is altered in cancer due to driver mutations in LINC proteins contributing to loss of epithelial polarity, aberrant tissue structure, abnormal gene expression, transformation and invasive cancer cell migration. The following aims are proposed: Aim 1. Define alterations to LINC complex- transmitted mechanical stresses in cancer. How the LINC complex transmits mechanical forces to position the nucleus and integrates tension in normal breast epithelia will be determined. The molecular and physical mechanisms for nuclear positioning in invasive breast epithelial cancer 3-D migration will be determined. Aim 2. Determine how the LINC complex contributes to altering the epigenetic organization of the genome during progression to breast cancer. The extent to which LINC disruption affects spatial partitioning of genes in the nucleus and heterochromatin organization will be identified; these effects will be correlated with cell phenotype, gene expression and epigenomic profiles. The requirement for an intact LINC complex for transformation to malignancy will be examined. The cancer nucleus remains highly understudied, with much to learn known about the physical principles that govern nuclear positioning, dysmorphia and chromatin organization, and how altered nuclear stresses contribute to cancer cell dysfunction. The focus of both aims is on the impact of cytoskeletal stresses transmitted by the LINC complex on gene expression and cell function. This necessarily requires an integrated understanding of both molecular and physical mechanisms. Extensive expertise gained in other systems will be coupled with new approaches for measuring forces on the nucleus. These include a direct force probe to interrogate nuclear mechanical responses in spread, living cells and nuclear tension sensors for the study of nuclear forces in both cancer and normal cells. Physically-based computational models will be used to interpret the resulting data. A physical approach will be applied to characterize the cancer nucleus and generate unique, genome-wide data sets for gene expression after experimentally altering LINC complex connections to discover the role of the LINC complex in breast cancer pathogenesis.